Method of treating chronic cardiac disease

ABSTRACT

New therapeutic uses for BPI protein products that involve treatment of chronic cardiac disease.

[0001] This application claims priority of U.S. provisional applicationSerial No. 60/116,736 filed Jan. 22, 1999, the disclosure of which isincorporated herein by reference.

[0002] The present invention relates generally to novel therapeutic usesof BPI protein products that involve treatment of chronic cardiacdisease including, but not limited to, chronic states such as congestiveheart failure and cardiomyopathy.

BACKGROUND OF THE INVENTION

[0003] Chronic cardiac disease is a leading cause of mortality andmorbidity in the developed world. One type of chronic cardiac disease iscardiomyopathy, which is actually a diverse group of diseasescharacterized by myocardial dysfunction that is not related to the usualcauses of heart disease such as coronary atherosclerosis, valvulardysfunction and hypertension. Cardiomyopathies are categorizedhemodynamically into dilated, hypertrophic, restrictive and obliterativecardiomyopathy, and can be of known or idiopathic etiology. Among theetiologies of dilated cardiomyopathy are pregnancy, drugs and toxins,such as alcohol, cocaine and chemotherapeutic agents (includingdoxorubicin and daunorubicin, dactinomycin, dacarbazine,cyclophosphamide, mitomycin, and anthracycline), and infectious andautoimmune processes. Hypertrophic cardiomyopathy is hereditary in morethan 50% of cases and has a distinctive pattern of myocardialhypertrophy (thickening of muscle) usually with a pattern ofasymmetrical thickening of the interventricular septum (also calledasymmetrical septal hypertrophy). Restrictive cardiomyopathies areusually the product of an infiltrative disease of the myocardium, suchas amyloidosis, hemochromatosis or a glycogen storage disease, and mayalso be seen in certain diabetic patients. Obliterative cardiomyopathycan be caused by endomyocardial fibrosis and hypereosinophilic syndrome.A common complication of all of the cardiomyopathies is progressivecongestive heart failure.

[0004] Congestive heart failure is often defined as the inability of theheart to deliver a supply of oxygenated blood sufficient to meet themetabolic needs of peripheral tissues at normal filling pressures.Chronic congestive heart failure can result as a consequence of coronaryartery disease, cardiomyopathy, myocarditis, aortic stenosis,hypertension, idiopathic asymmetrical septal hypertrophy, coarctation ofthe aorta, aortic regurgitation, mitral regurgitation, left-to-rightshunts, hypertrophied muscle, pericardial tamponade, constrictivepericarditis, mitral stenosis, left atrial mzxoma, left atrial thrombus,cor triatriatum and numerous other conditions. Congestive heart failureis generally distinguished from other causes of inadequate oxygendelivery, such as circulatory collapse from hemorrhage or other causesof severe volume loss, congestion caused by fluid overload andhigh-output failure caused by increased peripheral demands which occursin conditions such as thyrotoxicosis, arteriovenous fistula, Paget'sdisease and anemia. Therapy for congestive heart failure typicallyfocuses on the treating the underlying etiology and the symptoms offluid overload and heart failure. Chronic congestive heart failure thatpersists after correction of reversible causes is treated with diuretics(including thiazides such as chlorothiazide and hydrochlorothiazide,loop diuretics such as ethacrynic acid, furosemide, torsemide andbumetanide, potassium sparing agents such as spironolactone, triamtereneand amiloride, and others such as metolazone and otherquinazoline-sulfonamides), vasodilators (including nitroglycerin,isosorbide dinitrate, hydralazine, sodium nitroprusside, prostacyclin,captopril, enalapril, lisinopril, quinapril and losartan), positiveinotropic agents (such as digitalis or digoxin), occasionally betablockers, or combinations of these measures.

[0005] Recent studies indicate that an increase in pro-inflammatorycytokines is seen in diverse cardiac diseases, including congestiveheart failure, cardiomyopathy, and myocarditis. Hegewisch S, et al.Lancet 1990;2:294-295; Levine B, et al., N.Engl.J.Med. 1990;323(4):236-241; Mann D L, et al., Chest 1994;105:897-904; and Givertz M M,et al., Lancet 1998;352:34-38 For example, the cytokine tumor necrosisfactor-α (TNF) is synthesized by human cardiac myocytes, and the levelof TNF expression correlates with the degree of cardiac dysfunction inpatients. Torre-Amione G, et al., J.Am.Coll.Cardiol. 1996;27:1201-1206;Torre-Amione G, R D, et al. Circulation 1995;92:1487-1493; andTorre-Amione G, et al., Circulation 1996;93:704-711 In animals,synthesis of TNF by the heart is itself sufficient to causecardiomyopathy and lethal cardiac failure. Bryant D, et al., Circ.1998;97:175-183 and Kubota T, et al. J.Am.Coll.Cardiol.1997;346A(Abstract) Furthermore, early human trials have demonstratedthat antagonism of TNF improves cardiac failure in humans with NYHAClass III heart failure or idiopathic dilated cardiomyopathy. Deswal etal., Circulation 96: I-323 (1997); and Sliwa et al., Lancet 351:1091-1093 (1998) However, the primary stimulus for cytokine secretionremains unknown.

[0006] Bacterial endotoxin, or lipopolysaccharide (LPS), is a primaryinducer of TNF production during sepsis. With respect to cardiacdiseases, the role of endotoxin has been examined primarily in thecontext of cardiopulmonary bypass, driven by the hypothesis thatendotoxin may be present in the extracorporeal circuit, or may betranslocated across the intestine secondary to non-pulsatile, low flowperfusion. Riddington D W, et al. JAMA 1996;275:1007-1012 and Wan S, etal., Chest 1997;112:676-692 These studies have demonstrated onlytransient low-level endotoxemia during cardiopulmonary bypass, withrapid resolution following completion of cardiopulmonary bypass in themajority of patients. Nilsson L, J Thorac Cardiovasc Surg1990;100:777-780; Casey W F, Crit.Care Med. 1992;20 (8):1090-1096;Khabar K S, et al., Clin Immunol Immunopathol 1997;85:97-103; Jansen NJ, Ann Thorac Surg 1992;54:744-747. Bennett-Guerrero E et al., JAMA1997;277:646-650 reported that lower levels of anti-endotoxin antibodiespre-operatively were associated with an increased risk of post-operativecomplications and hypothesized that this difference was due to a poorimmunity to endotoxin.

[0007] Investigators have thus far failed to demonstrate, or failed toattempt to demonstrate, persistent endotoxemia in a majority of patientswith cardiac disease Nilsson L, J Thorac Cardiovasc Surg1990;100:777-780; Casey W F, Crit.Care Med. 1992;20 (8): 1090-1096;Khabar K S, et al., Clin Immunol Immunopathol 1997;85:97-103; Jansen NJ, Ann Thorac Surg 1992;54:744-747. See also Niebauer J, Eur. Heart J.1998;19:174, which reported elevated levels of plasma endotoxin inadults with edemetous chronic congestive heart failure that was notassociated with elevated levels of LBP or anti-endotoxin antibodies(indicators of long-term endotoxin exposure).

[0008] BPI is a protein isolated from the granules of mammalianpolymorphonuclear leukocytes (PMNs or neutrophils), which are bloodcells essential in the defense against invading microorganisms. HumanBPI protein has been isolated from PMNs by acid extraction combined witheither ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000(1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652(1987)]. BPI obtained in such a manner is referred to herein as naturalBPI and has been shown to have potent bactericidal activity against abroad spectrum of gram-negative bacteria. The molecular weight of humanBPI is approximately 55,000 daltons (55 kD). The amino acid sequence ofthe entire human BPI protein and the nucleic acid sequence of DNAencoding the protein have been reported in FIG. 1 of Gray et al., J.Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Grayet al. amino acid sequence is set out in SEQ ID NO: 1 hereto. U.S. Pat.No. 5,198,541 discloses recombinant genes encoding and methods forexpression of BPI proteins, including BPI holoprotein and fragments ofBPI.

[0009] BPI is a strongly cationic protein. The N-terminal half of BPIaccounts for the high net positive charge; the C-terminal half of themolecule has a net charge of −3. [Elsbach and Weiss (1981), supra.] Aproteolytic N-terminal fragment of BPI having a molecular weight ofabout 25 kD possesses essentially all the anti-bacterial efficacy of thenaturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio.Chem., 262: 14891-14894 (1987)]. In contrast to the N-terminal portion,the C-terminal region of the isolated human BPI protein displays onlyslightly detectable anti-bacterial activity against gram-negativeorganisms. [Ooi et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPIfragment of approximately 23 kD, referred to as “rBPI₂₃,” has beenproduced by recombinant means and also retains anti-bacterial activityagainst gram-negative organisms. [Gazzano-Santoro et al., Infect. Immun.60:4754-4761 (1992).] An N-terminal analog of BPI, rBPI₂₁, has beenproduced as described in Horwitz et al., Protein ExpressionPurification, 8:28-40 (1996).

[0010] The bactericidal effect of BPI was originally reported to behighly specific to gram-negative species, e.g., in Elsbach and Weiss,Inflammation: Basic Principles and Clinical Correlates, eds. Gallin etal., Chapter 30, Raven Press, Ltd. (1992). The precise mechanism bywhich BPI kills gram-negative bacteria is not yet completely elucidated,but it is believed that BPI must first bind to the surface of thebacteria through electrostatic and hydrophobic interactions between thecationic BPI protein and negatively charged sites on LPS. In susceptiblegram-negative bacteria, BPI binding is thought to disrupt LPS structure,leading to activation of bacterial enzymes that degrade phospholipidsand peptidoglycans, altering the permeability of the cell's outermembrane, and initiating events that ultimately lead to cell death.[Elsbach and Weiss (1992), supra]. LPS has been referred to as“endotoxin” because of the potent inflammatory response that itstimulates, i.e., the release of mediators by host inflammatory cellswhich may ultimately result in irreversible endotoxic shock. BPI bindsto lipid A, reported to be the most toxic and most biologically activecomponent of LPS.

[0011] BPI protein products have a wide variety of beneficialactivities. BPI protein products are bactericidal for gram-negativebacteria, as described in U.S. Pat. Nos. 5,198,541 and 5,523,288, bothof which are incorporated herein by reference. International PublicationNo. WO 94/20130 (incorporated herein by reference) proposes methods fortreating subjects suffering from an infection (e.g. gastrointestinal)with a species from the gram-negative bacterial genus Helicobacter withBPI protein products. BPI protein products also enhance theeffectiveness of antibiotic therapy in gram-negative bacterialinfections, as described in U.S. Pat. No. 5,523,288 and InternationalPublication No. WO 95/08344 (PCT/US94/11255), which are incorporatedherein by reference. BPI protein products are also bactericidal forgram-positive bacteria and mycoplasma, and enhance the effectiveness ofantibiotics in gram-positive bacterial infections, as described in U.S.Pat. Nos. 5,578,572 and 5,783,561 and International Publication No. WO95/19180 (PCT/US95/00656), which are incorporated herein by reference.BPI protein products exhibit anti-fungal activity, and enhance theactivity of other anti-fungal agents, as described in U.S. Pat. No.5,627,153 and International Publication No. WO 95/19179(PCT/US95/00498), and further as described for anti-fungal peptides inU.S. Pat. No. 5,858,974, which is in turn a continuation-in-part of U.S.application Ser. No. 08/504,841 filed Jul. 20, 1994 and correspondingInternational Publication Nos. WO 96/08509 (PCT/US95/09262) and WO97/04008 (PCT/US96/03845), all of which are incorporated herein byreference. BPI protein products exhibit anti-protozoan activity, asdescribed in U.S. Pat. No. 5,646,114 and International Publication No.WO 96/01647 (PCT/US95/08624), which are incorporated herein byreference. BPI protein products exhibit anti-chlamydial activity, asdescribed in co-owned, co-pending U.S. application Ser. No. 08/694,843filed Aug. 9, 1996 and WO 98/06415 (PCT/US97/13810), which areincorporated herein by reference. Finally, BPI protein products exhibitanti-mycobacterial activity, as described in co-owned, co-pending U.S.application Ser. No. 08/626,646 filed Apr. 1, 1996, which is in turn acontinuation of U.S. application Ser. No. 08/285,803 filed Aug. 14,1994, which is in turn a continuation-in-part of U.S. application Ser.No. 08/031,145 filed Mar. 12, 1993 and corresponding InternationalPublication No. WO94/20129 (PCT/US94/02463), all of which areincorporated herein by reference.

[0012] The effects of BPI protein products in humans with endotoxin incirculation, including effects on TNF, IL-6 and endotoxin are describedin U.S. Pat. No. 5,643,875, which is incorporated herein by reference.

[0013] BPI protein products are also useful for treatment of specificdisease conditions, such as meningococcemia in humans (as described inco-owned, co-pending U.S. application Ser. No. 08/644,287 filed May 10,1996 and continuation Ser. No. 08/927,437 filed Sep. 10, 1997 andInternational Publication No. WO97/42966 (PCT/US97/08016), all of whichare incorporated herein by reference), hemorrhagic trauma in humans, (asdescribed in U.S. Pat. No.5,756,464, U.S. application Ser. No.08/862,785 filed May 23, 1997 and corresponding InternationalPublication No. WO 97/44056 (PCT/US97/08941), all of which areincorporated herein by reference), burn injury (as described in U.S.Pat. No. 5,494,896, which is incorporated herein by reference),ischemia/reperfusion injury (as described in U.S. Pat. No. 5,578,568,incorporated herein by reference), and liver resection (as described inco-owned, co-pending U.S. application Ser. No. 08/582,230 filed Jan. 3,1996, which is in turn a continuation of U.S. application Ser. No.08/318,357 filed Oct. 5, 1994, which is in turn a continuation-in-partof U.S. application Ser. No. 08/132,510 filed Oct. 5, 1993, andcorresponding International Publication No. WO 95/10297(PCT/US94/11404), all of which are incorporated herein by reference).

[0014] BPI protein products also neutralize the anti-coagulant activityof exogenous heparin, as described in U.S. Pat. No. 5,348,942,incorporated herein by reference, and are useful for treating chronicinflammatory diseases such as rheumatoid and reactive arthritis and forinhibiting angiogenesis and for treating angiogenesis-associateddisorders including malignant tumors, ocular retinopathy andendometriosis, as described in U.S. Pat. Nos. 5,639,727, 5,807,818 and5,837,678 and International Publication No. WO 94/20128(PCT/US94/02401), all of which are incorporated herein by reference.

[0015] BPI protein products are also useful in antithrombotic methods,as described in U.S. Pat. No. 5,741,779 and U.S. application Ser. No.09/063,465 filed Apr. 20, 1998 and corresponding WO 97/42967(PCT/US7/08017), all of which are incorporated herein by reference.

SUMMARY OF THE INVENTION

[0016] The present invention provides novel therapeutic uses for BPIprotein products that involve treatment of subjects with chronic cardiacdisease. Uses of BPI protein products according to the invention arespecifically contemplated for prophylactic or therapeutic treatment ofchronic cardiac disease states or conditions in humans, particularlyhumans with chronic cardiac disease who exhibit elevated levels ofcirculating LPS and circulating LBP (in plasma or serum). Chroniccardiac disease states or conditions include but are not limited tocardiomyopathies, chronic congestive heart failure, and congenital heartdefects.

[0017] Chronic congestive heart failure as used herein includeslong-term congestive heart failure (i.e., congestive heart failurepersisting more than two weeks, or more than three weeks, or more thanone month, or more than two months, or more than three months),congestive heart failure that persists after correction of reversiblecauses, and congestive heart failure not immediately associated with anacute myocardial infarction or an acute infectious process.

[0018] Congenital heart defects, which may result in congestive heartfailure or cyanotic heart disease, include pulmonary atresia, totalanomalous pulmonary venous return, ventricular septal defect,hypoplastic left heart syndrome, double outlet right ventricle, rightpulmonary artery stenosis, interrupted aortic arch, Ebsteins's anomaly,tetralogy of Fallot, atrioventricular canal, transposition of the greatarteries and truncus arteriosus.

[0019] It is contemplated that the administration of a BPI proteinproduct may be accompanied by the concurrent administration of otherknown therapeutic agents for treating the chronic cardiac disease state.For example, agents that are known in the art for treating congestiveheart failure include diuretics (including thiazides such aschlorothiazide, hydrochlorothiazide and metolazone, loop diuretics suchas ethacrynic acid, furosemide, torsemide and bumetanide and theircongeners, potassium sparing agents such as spironolactone, canrenone,triamterene and amiloride, and others such as metolazone and otherquinazoline-sulfonamides), vasodilators (including nitrovasodilatorssuch as nitroglycerin, isosorbide dinitrate, and sodium nitroprusside,hydralazine, prostacyclin, ACE inhibitors such as captopril, enalapril,lisinopril, quinapril and ramipril, and angiotensin II antagonists suchas losartan), positive inotropic agents (such as cardiac glycosides,including digitalis or digoxin), phosphodiesterase inhibitors (such asamrinone and milrinone, primarily useful for short term support),occasionally beta-adrenergic receptor antagonists (beta blockers such aspropanolol, metoprolol, atenolol, pindolol, acebutolol, labetalol,carvedilol and celiprolol), or combinations of these measures. See,e.g., Goodman and Gilman, Ch. 34, The Pharmacological Basis ofTherapeutics, McGraw Hill, N.Y. (1996), incorporated by referenceherein.

[0020] The invention also contemplates use of a BPI protein product inthe preparation of a medicament for the prophylactic or therapeutictreatment of a chronic cardiac disease state.

[0021] Numerous additional aspects and advantages of the invention willbecome apparent to those skilled in the art upon consideration of thefollowing detailed description of the invention which describespresently preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1A displays plasma LPS levels for all patients completing thestudy protocol (n=29). FIG. 1B displays data from patients withendotoxemia pre-operatively (n=11), while FIG. 1C displays data frompatients without endotoxemia pre-operatively (n=18).

[0023]FIG. 2 displays plasma LBP levels from all patients completing thestudy protocol (n=29).

[0024]FIG. 3 displays plasma IL-6 levels from all patients completingthe study protocol (n=29).

[0025]FIGS. 4A and 4B displays pre-operative plasma LPS (FIG. 4B) andLBP (FIG. 4A) levels in patients with a severe (n=15), versus lesssevere (n=15), post-operative clinical course.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides novel therapeutic uses for BPIprotein products, particularly BPI-derived peptides, that involvetreatment of chronic cardiac disease. “Treatment” as used hereinencompasses both prophylactic and therapeutic treatment. The inventioncontemplates methods for treatment of subjects suffering from chronicheart disease which comprise the administration of therapeuticallyeffective amounts of bactericidal/permeability-increasing protein (BPI)protein products to those subjects so as to alleviate the negativephysiological effects of endotoxemia.

[0027] The invention is based on the discovery that a substantialproportion of subjects suffering from chronic heart disease exhibitevidence of endotoxemia associated with the chronic heart disease priorto surgery, and that this endotoxemia correlates to a poorer prognosisfor these subjects. Thus, one basis for the invention is the expectationthat endotoxemia is not simply a side effect of chronic cardiac diseasebut is a significant contributing factor to the pathology of chroniccardiac disease.

[0028] Therapeutic uses of BPI protein products are specificallycontemplated for treatment of mammals, including humans, suffering fromchronic cardiac disease as distinguished from acute cardiac diseasestates such as myocardial infarction, circulatory collapse fromhemorrhage and the like.

[0029] Another aspect of the present invention is the treatment ofpatients undergoing cardiopulmonary bypass, on the basis that theseverity of endotoxemia during and after cardiopulmonary bypass iscorrelated with poorer post-surgical outcome. Thus, treatment with BPIprotein product is expected to improve post-surgical outcome.

[0030] As used herein, “BPI protein product” includes naturally andrecombinantly produced BPI protein; natural, synthetic, and recombinantbiologically active polypeptide fragments of BPI protein; biologicallyactive polypeptide variants of BPI protein or fragments thereof,including hybrid fusion proteins and dimers; biologically activepolypeptide analogs of BPI protein or fragments or variants thereof,including cysteine-substituted analogs; and BPI-derived peptides. TheBPI protein products administered according to this invention may begenerated and/or isolated by any means known in the art. U.S. Pat. No.5,198,541, the disclosure of which is incorporated herein by reference,discloses recombinant genes encoding, and methods for expression of, BPIproteins including recombinant BPI holoprotein, referred to as rBPI andrecombinant fragments of BPI. U.S. Pat. No. 5,439,807 and correspondingInternational Publication No. WO 93/23540 (PCT/US93/04752), which areall incorporated herein by reference, disclose novel methods for thepurification of recombinant BPI protein products expressed in andsecreted from genetically transformed mammalian host cells in cultureand discloses how one may produce large quantities of recombinant BPIproducts suitable for incorporation into stable, homogeneouspharmaceutical preparations.

[0031] Biologically active fragments of BPI (BPI fragments) includebiologically active molecules that have the same or similar amino acidsequence as a natural human BPI holoprotein, except that the fragmentmolecule lacks amino-terminal amino acids, internal amino acids, and/orcarboxy-terminal amino acids of the holoprotein. Nonlimiting examples ofsuch fragments include an N-terminal fragment of natural human BPI ofapproximately 25 kD, described in Ooi et al., J. Exp. Med., 174:649(1991), and the recombinant expression product of DNA encodingN-terminal amino acids from 1 to about 193 to 199 of natural human BPI,described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992),and referred to as rBPI₂₃. In that publication, an expression vector wasused as a source of DNA encoding a recombinant expression product(rBPI₂₃) having the 31-residue signal sequence and the first 199 aminoacids of the N-terminus of the mature human BPI, as set out in FIG. 1 ofGray et al., supra, except that valine at position 151 is specified byGTG rather than GTC and residue 185 is glutamic acid (specified by GAG)rather than lysine (specified by AAG). Recombinant holoprotein (rBPI)has also been produced having the sequence (SEQ ID NOS: 1 and 2) set outin FIG. 1 of Gray et al., supra, with the exceptions noted for rBPI, andwith the exception that residue 417 is alanine (specified by GCT) ratherthan valine (specified by GTT). A fragment consisting of residues 10-193of BPI has been described in co-owned, co-pending U.S. application Ser.No. 09/099,725 filed Jun. 19, 1998, incorporated herein by reference.Other examples include dimeric forms of BPI fragments, as described inU.S. Pat. No. 5,447,913 and corresponding International Publication No.WO 95/24209 (PCT/US95/03125), all of which are incorporated herein byreference.

[0032] Biologically active variants of BPI (BPI variants) include butare not limited to recombinant hybrid fusion proteins, comprising BPIholoprotein or biologically active fragment thereof and at least aportion of at least one other polypeptide, and dimeric forms of BPIvariants. Examples of such hybrid fusion proteins and dimeric forms aredescribed in U.S. Pat. No. 5,643,570 and corresponding InternationalPublication No. WO 93/23434 (PCT/US93/04754), which are all incorporatedherein by reference and include hybrid fusion proteins comprising, atthe amino-terminal end, a BPI protein or a biologically active fragmentthereof and, at the carboxy-terminal end, at least one constant domainof an immunoglobulin heavy chain or allelic variant thereof.

[0033] Biologically active analogs of BPI (BPI analogs) include but arenot limited to BPI protein products wherein one or more amino acidresidues have been replaced by a different amino acid. For example, U.S.Pat. No. 5,420,019 and corresponding International Publication No. WO94/18323 (PCT/US94/01235), all of which are incorporated herein byreference, discloses polypeptide analogs of BPI and BPI fragmentswherein a cysteine residue is replaced by a different amino acid. Astable BPI protein product described by this application is theexpression product of DNA encoding from amino acid 1 to approximately193 or 199 of the N-terminal amino acids of BPI holoprotein, but whereinthe cysteine at residue number 132 is substituted with alanine and isdesignated rBPI₂₁Δcys or rBPI₂. Production of this N-terminal analog ofBPI, rBPI₂₁, has been described in Horwitz et al., Protein ExpressionPurification, 8:28-40 (1996). Similarly, a fragment consisting ofresidues 10-193 of BPI in which the cysteine at position 132 is replacedwith an alanine (designated “rBPI(10-193)C132A” or “rBPI(10-193)ala¹³²”)has been described in co-owned, co-pending U.S. application Ser. No.09/099,725 filed Jun. 19, 1998. Other examples include dimeric forms ofBPI analogs; e.g. U.S. Pat. No. 5,447,913 and correspondingInternational Publication No. WO 95/24209 (PCT/US95/03125), all of whichare incorporated herein by reference.

[0034] Other BPI protein products useful according to the methods of theinvention are peptides derived from or based on BPI produced bysynthetic or recombinant means (BPI-derived peptides), such as thosedescribed in International Publication No. WO 97/04008 (PCT/US96/03845),which corresponds to U.S. application Ser. No. 08/621,259 filed Mar. 21,1996, and International Publication No. WO 96/08509 (PCT/US95/09262),which corresponds to U.S. Pat. No. 5,858,974, and InternationalPublication No. WO 95/19372 (PCT/US94/10427), which corresponds to U.S.Pat. No. 5,652,332, and International Publication No. WO94/20532(PCT/US94/02465), which corresponds to U.S. Pat. No. 5,763,567 which isa continuation of U.S. Pat. No. 5,733,872, which is acontinuation-in-part of U.S. application Ser. No. 08/183,222, filed Jan.14, 1994, which is a continuation-in-part of U.S. application Ser. No.08/093,202 filed Jul. 15, 1993 (corresponding to InternationalPublication No. WO 94/20128 (PCT/US94/02401)), which is acontinuation-in-part of U.S. Pat. No. 5,348,942, as well asInternational Application No. PCT/US97/05287, which corresponds to U.S.Pat. No. 5,851,802, the disclosures of all of which are incorporatedherein by reference.

[0035] Presently preferred BPI protein products includerecombinantly-produced N-terminal analogs and fragments of BPI,especially those having a molecular weight of approximately between 20to 25 kD such as rBPI₂₁ or rBPI₂₃, rBPI(10-193)C132A(rBPI(10-193)ala¹³²), dimeric forms of these N-terminal proteins (e.g.,rBPI₄₂ dimer), and BPI-derived peptides.

[0036] The administration of BPI protein products is preferablyaccomplished with a pharmaceutical composition comprising a BPI proteinproduct and a pharmaceutically acceptable diluent, adjuvant, or carrier.The BPI protein product may be administered without or in conjunctionwith known surfactants or other therapeutic agents. A stablepharmaceutical composition containing BPI protein products (e.g.,rBPI₂₃) comprises the BPI protein product at a concentration of 1 mg/mlin citrate buffered saline (5 or 20 mM citrate, 150 mM NaCl, pH 5.0)comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASFWyandotte, Parsippany, N.J.) and 0.002% by weight of polysorbate 80(Tween 80, ICI Americas Inc., Wilmington, Del.). Another stablepharmaceutical composition containing BPI protein products (e.g.,rBPI₂₁) comprises the BPI protein product at a concentration of 2 mg/mlin 5 mM citrate, 150 mM NaCl, 0.2% poloxamer 188 and 0.002% polysorbate80. Such preferred combinations are described in U.S. Pat. Nos.5,488,034 and 5,696,090 and corresponding International Publication No.WO 94/17819 (PCT/US94/01239), the disclosures of all of which areincorporated herein by reference. As described in U.S. application Ser.No. 08/586,133 filed Jan. 12, 1996, which is in turn acontinuation-in-part of U.S. application Ser. No. 08/530,599 filed Sep.19, 1995, which is in turn a continuation-in-part of U.S. applicationSer. No. 08/372,104 filed Jan. 13, 1995, and corresponding InternationalPublication No. WO96/21436 (PCT/US96/01095), all of which areincorporated herein by reference, other poloxamer formulations of BPIprotein products with enhanced activity may be utilized.

[0037] Therapeutic compositions comprising BPI protein product may beadministered systemically or topically. Systemic routes ofadministration include oral, intravenous, intramuscular or subcutaneousinjection (including into a depot for long-term release), intraocularand retrobulbar, intrathecal, intraperitoneal (e.g. by intraperitoneallavage), intrapulmonary (using powdered drug, or an aerosolized ornebulized drug solution), or transdermal.

[0038] When given parenterally, BPI protein product compositions aregenerally injected in doses ranging from 1 μg/kg to 100 mg/kg per day,preferably at doses ranging from 0.1 mg/kg to 20 mg/kg per day, morepreferably at doses ranging from 1 to 20 mg/kg/day and most preferablyat doses ranging from 2 to 10 mg/kg/day. The treatment may continue bycontinuous infusion or intermittent injection or infusion, at the same,reduced or increased dose per day for, e.g., 1 to 3 days, andadditionally as determined by the treating physician. When administeredintravenously, BPI protein products are preferably administered by aninitial brief infusion followed by a continuous infusion. The preferredintravenous regimen is a 1 to 20 mg/kg brief intravenous infusion of BPIprotein product followed by a continuous intravenous infusion at a doseof 1 to 20 mg/kg/day, continuing for up to one week. A particularlypreferred intravenous dosing regimen is a 1 to 4 mg/kg initial briefintravenous infusion followed by a continuous intravenous infusion at adose of 1 to 4 mg/kg/day, continuing for up to 72 hours.

[0039] Those skilled in the art can readily optimize effective dosagesand administration regimens for therapeutic compositions comprising BPIprotein product, as determined by good medical practice and the clinicalcondition of the individual patient.

[0040] “Concurrent administration,” or “co-administration,” as usedherein includes administration of the agents, in conjunction orcombination, together, or before or after each other. The BPI proteinproduct and second agent(s) may be administered by different routes. Forexample, the BPI protein product may be administered intravenously whilethe second agent(s) is(are) administered intravenously, intramuscularly,subcutaneously, orally or intraperitoneally. The BPI protein product andsecond agent(s) may be given sequentially in the same intravenous lineor may be given in different intravenous lines. Alternatively, the BPIprotein product may be administered in a special form for gastricdelivery, while the second agent(s) is(are) administered, e.g., orally.The formulated BPI protein product and second agent(s) may beadministered simultaneously or sequentially, as long as they are givenin a manner sufficient to allow all agents to achieve effectiveconcentrations at the site of action.

[0041] Other aspects and advantages of the present invention will beunderstood upon consideration of the following illustrative examples.Example 1 addresses a study in which thirty children with complexchronic heart disease were tested for markers of endotoxemia prior toand at 1, 8, 24, 48 and 72 hours following cardiopulmonary bypasssurgery.

EXAMPLE 1

[0042] The experimental protocol, approved by the Institutional ReviewBoard at the University of Texas Southwestern Medical Center, was anunblinded, prospective study in which 30 children with severe congenitalheart disease were sequentially enrolled while awaiting surgical repairand/or palliation. One patient with hypoplastic left heart syndrome diedintra-operatively, and therefore data on this child are included only inthe pre-operative analysis. Patients with clinical evidence ofpreoperative infection were excluded from the study.

[0043] The 30 enrolled children ranged in age from 4 days to 402 days(median age 59 days), and in weight from 2.0 to 9.5 kg (median weight4.0 kg) The genders, ages, cardiac diagnoses, and surgical repairs arelisted in Table 1 below. TABLE 1 Patient Characteristics Age- Sex davsWt (kg) Diagnosis* Procedure M 349 8.90 Pulmonary Atresia RVOTReconstruction M 6 5.70 TAPVR TAPVR Repair F 265 6.60 TAPVR TAPVR RepairM 5 3.30 Interrupted Aortic Arch Aortic Arch Repair M 44 3.50 Ebstein'sanomaly, VSD VSD Repair M 245 8.09 VSD VSD Repair F 7 2.50 HLHS NorwoodProcedure F 10 3.4 HLHS Norwood Procedure F 210 6.70 VSD VSD Repair M163 5.00 DORV, RPA Stenosis DKS F 395 8.20 TOF Tetralogy Repair M 102.50 TGA, DORV Arterial Switch M 75 3.60 AV Canal AV Canal Repair M 44.40 TGA Arterial Switch M 4 3.60 TGA Arterial Switch M 4 3.35 LVRhabdomyosarcoma Tumor Resection M 26 3.70 TAPVR TAPVR Repair F 110 3.20TAPVR TAPVR Repair F 126 2.00 Truncus Arteriosus RVOT Reconstruction M402 9.50 TOF Tetralogy Repair M 25 2.60 Pulmonary Atresia RVOTReconstruction F 105 4.40 AV Canal AV Canal Repair M 178 5.80 TOFTetralogy Repair M 5 4.40 TGA Arterial Switch M 102 4.10 TAPVR TAPVRRepair F 5 3.70 TAPVR TAPVR Repair M 6 3.10 HLHS Norwood Procedure F 1205.40 AV Canal AV Canal Repair M 4 3.80 TGA Arterial Switch M 335 7.50TOF Tetralogy Repair

[0044] Anesthesia was induced with sevoflurane, nitrous oxide andoxygen; intubation was facilitated with intravenous rocuronium andfentanyl. Anesthesia was maintained with fentanyl (30-50 mcg/kg),isoflurane, and pancuronium. Nine patients received tranexamic acid(50-100 mg/kg) and 3 received aprotinin (dosed to achieve 350 units/mLtotal blood volume).

[0045] Cardiopulmonary bypass was performed as follows. Theextracorporeal circuit consisted of a roller pump, membrane oxygenator,and cardiotomy filters Prior to the institution of cardiopulmonarybypass, the patients' blood was anticoagulated with heparin (300units/kg). 13 patients underwent deep hypothermic circulatory arrest(core temp 16-18° C.) and the remainder were cooled to a coretemperature of 25-30° C. for the completion of surgery. Hemofiltrationwas performed prior to completion of cardiopulmonary bypass on allpatients in an attempt to remove excess free water and attain ahemoglobin >12 gm/dL.

[0046] Blood samples for the determination of LPS, LBP, and IL-6 wereobtained prior to surgery and at 1, 8, 24, 48 and 72 hours aftercompletion of cardiopulmonary bypass. The pre-operative sample wasobtained from a newly placed central venous catheter, immediately afterthe induction of anesthesia and endotracheal intubation. Fordetermination of endotoxin levels, blood samples were collected intoheparinized Vacutainer™ tubes (Becton-Dickson, Rutherford N.J.) selectedfor low endotoxin content (BioWhitaker, Walkersville, Md.), immediatelyplaced on ice, and walked to the laboratory by an investigator.Platelet-rich plasma was obtained by centrifugation (180×g, 10 min, 2-8°C.). Samples were stored at −70° C. until assay.

[0047] LPS, LBP and IL-6 assays were conducted in a blinded fashion. Thelevel of LPS in the platelet-rich plasma was determined by using akinetic chromogenic Limulus amebocyte lysate assay (Endochrome-K™,Endosafe, Charleston, S.C.) according to the manufacturer'sinstructions. LPS concentrations are expressed in terms of endotoxinunits (EU) per ml relative to an E. coli O55:B5 control standardendotoxin. LBP levels were determined by ELISA as described in MeszarosS, et al., Infect.Immun. 1995;63:363-365. IL-6 was measured using asandwich ELISA (R&D Systems, Minneapolis).

[0048] A severe (versus less-severe) post operative course wasprospectively defined. Severity of post-operative myocardial dysfunctionwas estimated according to an adaptation of the scale utilized byWernovsky et al., Circulation 1995;92:2226-2235. Specifically, aninotropic support score was calculated as follows: each 1.0 mcg/kg/minof dopamine or dobutamine, and each 0.01 mcg/kg/min of epinephrineyielded a score of 1. Children with a net positive fluid balance of >40cc/kg in the first 24 hours and an inotropic support score of >12, orperioperative death, were considered to have a severe clinical course.Post-operative severity of illness was scored prior to knowledge of LPS,LBP, or IL-6 values.

[0049] All statistical analyses were performed on the StatisticalPackage for the Social Sciences (SPSS). Wilcoxon signed rank tests fornon-parametric data were performed to determine if a significant rise inLPS, LBP. or IL-6 had occurred post-operatively. A Mann-Whitney test fornon-parametric data was performed to determine if there was asignificant difference in LPS or LBP concentrations between the patientswho had a more severe clinical course compared to those with a lesssevere clinical course.

[0050] Twenty-nine of the thirty patients (96%) had evidence ofendotoxemia during the study period, either by detection of elevated LPSdirectly or by detection of an elevated LBP plasma level >2SD above themean for healthy adults. The LPS, LBP and IL-6 levels for all patientsare displayed in FIGS. 1A, 2 and 3. FIG. 1A displays data from allchildren completing the study protocol and demonstrate elevated LPS atall time points. Differences between pre-operative and post-operativeLPS levels are statistically non-significant. To better elucidateendotoxin kinetics, we divided patients into two groups: those who wereendotoxemic prior to cardiopulmonary bypass, and those who were notendotoxemic prior to cardiopulmonary bypass (CPB). Levels for these twogroups are shown in FIGS. 1B and 1C, respectively.

[0051] Prior to CPB, 12 patients had significant elevation of plasmaendotoxin. In these patients, endotoxin tended to decline followingcompletion of cardiopulmonary bypass, likely due to hemodilution/partialexchange transfusion (FIG. 1B); but endotoxin levels remained abnormallyelevated throughout the study period. In those children withoutpre-operative endotoxemia, the level of plasma endotoxin rosesignificantly following bypass, achieving a peak value at 1 hour postbypass, and remaining significantly elevated thereafter (p<0.0001) (FIG.1C).

[0052] There was a transient, but significant decrease in plasma LBPimmediately after completion of cardiopulmonary bypass andhemofiltration. The decrease in plasma LBP at 1 hour followingcardiopulmonary bypass was statistically significant (p<0.0001), and theincrease in LBP at all points thereafter was also highly consistent andstatistically significant (p<0.0001) compared to preoperative levels.This rise in LBP was similar for patients who were and were notendotoxemic preoperatively. Similarly, there was a significant rise inIL-6 at all time points following CPB (p<0.05) compared to preoperativelevels (FIG. 3).

[0053] Finally, it was determined whether children who experienced amore severe clinical course, defined prospectively, might differ fromless severe patients when pre-operative LPS and LBP levels werecompared. In this comparison, the more severely ill children hadsignificantly higher pre-operative plasma LBP (p<0.02) (FIG. 4A), andtended toward higher pre-operative LPS (p<0.05) compared to patients whoexperienced a less severe post-operative course (FIG. 4B). Additionally,of the 12 patients who were endotoxemic prior to surgery, there were 3deaths (25%), compared to 0 deaths in the 18 patients who were notendotoxemic prior to surgery (p=0.054).

[0054] The underlying biology of peri-operative endotoxemia is clarifiedby dividing those patients who were or were not endotoxemic-preoperatively. In patients who were endotoxemic pre-operatively, endotoxinlevels initially fell following cardiopulmonary bypass, but remainedabnormally elevated throughout the study period. This initial decreasemay have been secondary to a dilution effect of cardiopulmonary bypass,given the infants' small blood volumes, or perhaps due to clearance ofendotoxin by hemofiltration prior to completion of cardiopulmonarybypass. Millar A B, Ann Thorac Surg 1993;56:1499-1502 It is alsopossible that these patients, who were endotoxemic pre-operatively, mayhave induced and enhanced mechanisms for endotoxin clearance, comparedto patients who were not endotoxemic pre-operatively. Dentener M A, etal.,. Journal of Infectious Diseases 1997;175:108-117 Dentener M A,Journal of Infectious Diseases 1995;171:739-743; and Gazzano-Santoro etal., Infect.Immunol. 1994;62, No4:1185-1191

[0055] In contrast, patients who were not endotoxemic pre-operativelydemonstrated a significant elevation of plasma endotoxin at one andeight hours after cardiopulmonary bypass. A number of factors have beeninvoked to explain endotoxemia during cardiopulmonary bypass. First,there are many sources of endotoxin including the extracorporealcircuit, infusion solutions, drugs, and surgical materials. Moreimportantly, increased intestinal permeability during cardiopulmonarybypass has been documented in adult patients, allowing for bacterialtranslocation and release of endotoxin into the circulation. Measuressuch as pulsatile perfusion or higher flow during bypass to improve gutperfusion and aggressive antibiotic regimens to decrease intestinalbacterial load prior to bypass have resulted in lower plasma LPS level.Watarida S, et al., J Thorac Cardiovasc Surg 1994;108:620-625; andQuigley R L, et al. Perfusion 1995;10:27-31.

[0056] The finding of elevated LPS and LBP levels pre-operatively in asubstantial proportion of children with chronic cardiac diseaseindicates that endotoxemia is associated with the chronic cardiacdisease itself. The finding that severity of outcome correlated withendotoxemia as measured by elevated LPS and LBP levels indicates thatmeasurement of endotoxemia can be used to predict prognosis.Consequently, the treatment of endotoxemia with BPI protein product isexpected to ameliorate the signs and symptoms of chronic cardiac diseaseand to improve the prognosis of these patients.

[0057] Numerous modifications and variations of the above-describedinvention are expected to occur to those of skill in the art.Accordingly, only such limitations as appear in the appended claimsshould be placed thereon.

1 2 1 1813 DNA Homo sapiens CDS (31)..(1491) mat_peptide (124)..(1491) 1caggccttga ggttttggca gctctggagg atg aga gag aac atg gcc agg ggc 54 MetArg Glu Asn Met Ala Arg Gly -30 -25 cct tgc aac gcg ccg aga tgg gtg tccctg atg gtg ctc gtc gcc ata 102 Pro Cys Asn Ala Pro Arg Trp Val Ser LeuMet Val Leu Val Ala Ile -20 -15 -10 ggc acc gcc gtg aca gcg gcc gtc aaccct ggc gtc gtg gtc agg atc 150 Gly Thr Ala Val Thr Ala Ala Val Asn ProGly Val Val Val Arg Ile -5 -1 1 5 tcc cag aag ggc ctg gac tac gcc agccag cag ggg acg gcc gct ctg 198 Ser Gln Lys Gly Leu Asp Tyr Ala Ser GlnGln Gly Thr Ala Ala Leu 10 15 20 25 cag aag gag ctg aag agg atc aag attcct gac tac tca gac agc ttt 246 Gln Lys Glu Leu Lys Arg Ile Lys Ile ProAsp Tyr Ser Asp Ser Phe 30 35 40 aag atc aag cat ctt ggg aag ggg cat tatagc ttc tac agc atg gac 294 Lys Ile Lys His Leu Gly Lys Gly His Tyr SerPhe Tyr Ser Met Asp 45 50 55 atc cgt gaa ttc cag ctt ccc agt tcc cag ataagc atg gtg ccc aat 342 Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln Ile SerMet Val Pro Asn 60 65 70 gtg ggc ctt aag ttc tcc atc agc aac gcc aat atcaag atc agc ggg 390 Val Gly Leu Lys Phe Ser Ile Ser Asn Ala Asn Ile LysIle Ser Gly 75 80 85 aaa tgg aag gca caa aag aga ttc tta aaa atg agc ggcaat ttt gac 438 Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Met Ser Gly AsnPhe Asp 90 95 100 105 ctg agc ata gaa ggc atg tcc att tcg gct gat ctgaag ctg ggc agt 486 Leu Ser Ile Glu Gly Met Ser Ile Ser Ala Asp Leu LysLeu Gly Ser 110 115 120 aac ccc acg tca ggc aag ccc acc atc acc tgc tccagc tgc agc agc 534 Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser SerCys Ser Ser 125 130 135 cac atc aac agt gtc cac gtg cac atc tca aag agcaaa gtc ggg tgg 582 His Ile Asn Ser Val His Val His Ile Ser Lys Ser LysVal Gly Trp 140 145 150 ctg atc caa ctc ttc cac aaa aaa att gag tct gcgctt cga aac aag 630 Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser Ala LeuArg Asn Lys 155 160 165 atg aac agc cag gtc tgc gag aaa gtg acc aat tctgta tcc tcc aag 678 Met Asn Ser Gln Val Cys Glu Lys Val Thr Asn Ser ValSer Ser Lys 170 175 180 185 ctg caa cct tat ttc cag act ctg cca gta atgacc aaa ata gat tct 726 Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Met ThrLys Ile Asp Ser 190 195 200 gtg gct gga atc aac tat ggt ctg gtg gca cctcca gca acc acg gct 774 Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pro ProAla Thr Thr Ala 205 210 215 gag acc ctg gat gta cag atg aag ggg gag ttttac agt gag aac cac 822 Glu Thr Leu Asp Val Gln Met Lys Gly Glu Phe TyrSer Glu Asn His 220 225 230 cac aat cca cct ccc ttt gct cca cca gtg atggag ttt ccc gct gcc 870 His Asn Pro Pro Pro Phe Ala Pro Pro Val Met GluPhe Pro Ala Ala 235 240 245 cat gac cgc atg gta tac ctg ggc ctc tca gactac ttc ttc aac aca 918 His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp TyrPhe Phe Asn Thr 250 255 260 265 gcc ggg ctt gta tac caa gag gct ggg gtcttg aag atg acc ctt aga 966 Ala Gly Leu Val Tyr Gln Glu Ala Gly Val LeuLys Met Thr Leu Arg 270 275 280 gat gac atg att cca aag gag tcc aaa tttcga ctg aca acc aag ttc 1014 Asp Asp Met Ile Pro Lys Glu Ser Lys Phe ArgLeu Thr Thr Lys Phe 285 290 295 ttt gga acc ttc cta cct gag gtg gcc aagaag ttt ccc aac atg aag 1062 Phe Gly Thr Phe Leu Pro Glu Val Ala Lys LysPhe Pro Asn Met Lys 300 305 310 ata cag atc cat gtc tca gcc tcc acc ccgcca cac ctg tct gtg cag 1110 Ile Gln Ile His Val Ser Ala Ser Thr Pro ProHis Leu Ser Val Gln 315 320 325 ccc acc ggc ctt acc ttc tac cct gcc gtggat gtc cag gcc ttt gcc 1158 Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val AspVal Gln Ala Phe Ala 330 335 340 345 gtc ctc ccc aac tcc tcc ctg gct tccctc ttc ctg att ggc atg cac 1206 Val Leu Pro Asn Ser Ser Leu Ala Ser LeuPhe Leu Ile Gly Met His 350 355 360 aca act ggt tcc atg gag gtc agc gccgag tcc aac agg ctt gtt gga 1254 Thr Thr Gly Ser Met Glu Val Ser Ala GluSer Asn Arg Leu Val Gly 365 370 375 gag ctc aag ctg gat agg ctg ctc ctggaa ctg aag cac tca aat att 1302 Glu Leu Lys Leu Asp Arg Leu Leu Leu GluLeu Lys His Ser Asn Ile 380 385 390 ggc ccc ttc ccg gtt gaa ttg ctg caggat atc atg aac tac att gta 1350 Gly Pro Phe Pro Val Glu Leu Leu Gln AspIle Met Asn Tyr Ile Val 395 400 405 ccc att ctt gtg ctg ccc agg gtt aacgag aaa cta cag aaa ggc ttc 1398 Pro Ile Leu Val Leu Pro Arg Val Asn GluLys Leu Gln Lys Gly Phe 410 415 420 425 cct ctc ccg acg ccg gcc aga gtccag ctc tac aac gta gtg ctt cag 1446 Pro Leu Pro Thr Pro Ala Arg Val GlnLeu Tyr Asn Val Val Leu Gln 430 435 440 cct cac cag aac ttc ctg ctg ttcggt gca gac gtt gtc tat aaa 1491 Pro His Gln Asn Phe Leu Leu Phe Gly AlaAsp Val Val Tyr Lys 445 450 455 tgaaggcacc aggggtgccg ggggctgtcagccgcacctg ttcctgatgg gctgtggggc 1551 accggctgcc tttccccagg gaatcctctccagatcttaa ccaagagccc cttgcaaact 1611 tcttcgactc agattcagaa atgatctaaacacgaggaaa cattattcat tggaaaagtg 1671 catggtgtgt attttaggga ttatgagcttctttcaaggg ctaaggctgc agagatattt 1731 cctccaggaa tcgtgtttca attgtaaccaagaaatttcc atttgtgctt catgaaaaaa 1791 aacttctggt ttttttcatg tg 1813 2487 PRT Homo sapiens 2 Met Arg Glu Asn Met Ala Arg Gly Pro Cys Asn AlaPro Arg Trp Val -30 -25 -20 Ser Leu Met Val Leu Val Ala Ile Gly Thr AlaVal Thr Ala Ala Val -15 -10 -5 -1 1 Asn Pro Gly Val Val Val Arg Ile SerGln Lys Gly Leu Asp Tyr Ala 5 10 15 Ser Gln Gln Gly Thr Ala Ala Leu GlnLys Glu Leu Lys Arg Ile Lys 20 25 30 Ile Pro Asp Tyr Ser Asp Ser Phe LysIle Lys His Leu Gly Lys Gly 35 40 45 His Tyr Ser Phe Tyr Ser Met Asp IleArg Glu Phe Gln Leu Pro Ser 50 55 60 65 Ser Gln Ile Ser Met Val Pro AsnVal Gly Leu Lys Phe Ser Ile Ser 70 75 80 Asn Ala Asn Ile Lys Ile Ser GlyLys Trp Lys Ala Gln Lys Arg Phe 85 90 95 Leu Lys Met Ser Gly Asn Phe AspLeu Ser Ile Glu Gly Met Ser Ile 100 105 110 Ser Ala Asp Leu Lys Leu GlySer Asn Pro Thr Ser Gly Lys Pro Thr 115 120 125 Ile Thr Cys Ser Ser CysSer Ser His Ile Asn Ser Val His Val His 130 135 140 145 Ile Ser Lys SerLys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 150 155 160 Ile Glu SerAla Leu Arg Asn Lys Met Asn Ser Gln Val Cys Glu Lys 165 170 175 Val ThrAsn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu 180 185 190 ProVal Met Thr Lys Ile Asp Ser Val Ala Gly Ile Asn Tyr Gly Leu 195 200 205Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Leu Asp Val Gln Met Lys 210 215220 225 Gly Glu Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe Ala Pro230 235 240 Pro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val Tyr LeuGly 245 250 255 Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Leu Val Tyr GlnGlu Ala 260 265 270 Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met Ile ProLys Glu Ser 275 280 285 Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Thr PheLeu Pro Glu Val 290 295 300 305 Ala Lys Lys Phe Pro Asn Met Lys Ile GlnIle His Val Ser Ala Ser 310 315 320 Thr Pro Pro His Leu Ser Val Gln ProThr Gly Leu Thr Phe Tyr Pro 325 330 335 Ala Val Asp Val Gln Ala Phe AlaVal Leu Pro Asn Ser Ser Leu Ala 340 345 350 Ser Leu Phe Leu Ile Gly MetHis Thr Thr Gly Ser Met Glu Val Ser 355 360 365 Ala Glu Ser Asn Arg LeuVal Gly Glu Leu Lys Leu Asp Arg Leu Leu 370 375 380 385 Leu Glu Leu LysHis Ser Asn Ile Gly Pro Phe Pro Val Glu Leu Leu 390 395 400 Gln Asp IleMet Asn Tyr Ile Val Pro Ile Leu Val Leu Pro Arg Val 405 410 415 Asn GluLys Leu Gln Lys Gly Phe Pro Leu Pro Thr Pro Ala Arg Val 420 425 430 GlnLeu Tyr Asn Val Val Leu Gln Pro His Gln Asn Phe Leu Leu Phe 435 440 445Gly Ala Asp Val Val Tyr Lys 450 455

What is claimed is:
 1. A method of treating a human with chronic cardiacdisease comprising the step of administering a therapeutically effectiveamount of a bactericidal/permeability-increasing (BPI) protein productto said human.
 2. The method of claim 1 wherein the BPI protein productis rBPI₂₁.
 3. The method of claim 1 wherein the chronic cardiac diseaseis chronic congestive heart failure.
 4. The method of claim 1 whereinthe chronic cardiac disease is cardiomyopathy.
 5. The method of claim 1wherein the chronic cardiac disease is a congenital heart defect.
 6. Themethod of claim 1 wherein the human exhibits elevated levels ofcirculating LPS.
 7. The method of claim 1 wherein the human exhibitselevated levels of circulating LBP.
 8. The method of claim 1 wherein thehuman exhibits elevated levels of circulating LPS and circulating LBP.9. The method of claim 1 further comprising concurrently administering asecond therapeutic agent for treating the chronic cardiac disease state.10. The method of claim 9 wherein said chronic cardiac disease state ischronic congestive heart failure and the second therapeutic agent isselected from the group consisting of diuretics, positive inotropicagents, vasodilators and beta-blockers.